Method of lightening skin

ABSTRACT

A method and composition for the topical application of a yohimbine or derivative or stereoisomer thereof or effective α 2  -antagonists, to the skin for inhibiting melanogenesis in the skin leading to skin lightening. The yohimbine may be provided as a natural extract, for example, as an extract of Johimbe bark.

BACKGROUND

The present invention generally relates to depigmenting orskin-lightening compositions for topical application on a subject's skinand methods of their use.

Skin color in humans arises from a complex series of cellular processeswhich are carried out within a unique population of cells calledmelanocytes. Melanocytes are located in the lower part of the epidermis,and their function is to synthesize a brown pigment, melanin, whichprotects the body from the damaging effects of ultraviolet radiation.Melanin is deposited in melanosomes, which are vesicles found within themelanocytes. The melanosomes are extruded from the melanocytes andcarried to the surface of the skin by keratinocytes, which internalizethe melanin containing melanosomes. The darkness of the color observedin the skin is proportionate to the amount of melanin synthesized bymelanocytes and transferred to the keratinocytes. In some cases, it isdesirable to reduce or inhibit melanogenesis, for example, to cause skinlightening, to eliminate "age spots" or to reduce hyperactivemelanocytes.

SUMMARY OF THE INVENTION

The objective of the present invention is to provide a method andcomposition for topical application to the skin for inhibitingmelanogenesis thereby causing skin lightening.

In a preferred version the present invention comprises a composition ofmatter comprising an amount of an active agent comprising yohimbine oran effective derivative, stereoisomer or salt thereof effective indecreasing levels of melanin in a human melanocyte. The composition alsocomprises an effective amount of a pharmaceutically acceptable topicalcarrier which is capable of delivering the yohimbine or stereoisomer,derivative or salt thereof to the melanocyte-containing skin layer underin vivo conditions. The active agent may be provided as a naturalextract, for example, as an extract of Johimbe tree bark.

The present invention also comprises a method for decreasingpigmentation or promoting lightening of the skin of a subject. Thesubject's skin is treated with a depigmenting composition comprising ayohimbine effective in decreasing the amount of melanin in a humanmelanocyte, and a pharmaceutically acceptable topical carrier aspreviously described. Such a method of application may further comprisea sunscreen composition to at least partially shield the subject's skinfrom ultraviolet radiation.

More particularly, the present invention contemplates a composition, andmethod of using such, which is effective in inhibiting melanogenesis inhuman skin, the composition comprising an effective amount of ayohimbine capable of decreasing levels of melanin in melanocytes inhuman skin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the dose response effect of yohimbine ontyrosinase activity in melanocytes.

FIG. 2 is a graph showing the effect of free base yohimbine ontyrosinase activity in melanocytes.

FIG. 3 is a graph showing the comparative effects of yohimbine and theyohimbine stereoisomer rauwolscine on tyrosinase activity inmelanocytes.

FIG. 4 is a photograph and graph showing the lightening effect ofyohimbine on a human skin culture.

FIG. 5 is a graph showing the recovery of tyrosinase activity in humanmelanocyte cell culture after removal of yohimbine from the culturemedium.

FIG. 6 is a graph showing the effects of the α₂ -antagonists BU224 andefaroxan on tyrosinase activity in another human melanocyte cellculture.

FIG. 7 is a graph showing the effects of phentolamine and the yohimbinestereoisomer corynanthine on tyrosinase activity in another humanmelanocyte cell culture.

FIG. 8 is a photograph and graph showing the depigmentizing effect ofnatural Johimbe bark extract on a human skin culture.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to compositions and methods whichdecrease melanogenesis in the skin of subjects. "Subjects" as usedherein mean mammals, and, more preferably, humans.

The present invention comprises compositions comprising an effectiveamount of one or more yohimbines or active agents (e.g., α₂-antagonists) capable of decreasing the amount of melanin in amelanocyte (also referred to herein as "melanin-decreasing agents") andpreferably, in a human melanocyte, and more preferably, in melanocytesin intact human skin. The melanin-decreasing agent may function in anyof the various ways that are believed to decrease the amount of melaninproduced within the skin, for example, by decreasing cAMP (cyclic AMP)or derivatives of cAMP which function as cAMP in the melanocyte; bypromoting phosphodiesterase; or by decreasing tyrosinase activity. Thepresent invention, as noted above, additionally comprises all α₂-antagonists which are effective in accordance with the presentinvention wherein the melanogenesis in melanocytes is inhibited leadingto a lightening of the skin.

It is Applicant's unique discovery therefore, described further herein,that yohimbines, and certain other agents (such as certain α₂-antagonists) are effective in decreasing melanin production inmelanocytes thereby reducing skin pigmentation resulting in skinlightening.

More particularly, the present invention contemplates a composition, andmethod of using such, which is effective in inhibiting melanogenesis inhuman skin. The composition comprises an effective amount of a yohimbinecapable of decreasing levels of melanin in a human melanocyte. Whereused herein the term yohimbine is meant to include stereoisomers, salts,and derivatives of yohimbine which are also effective in depigmentation.Examples are rauwolscine (α-yohimbine), allo-yohimbine, and corynanthine(rauhimbine).

The yohimbine or other active agent as described herein may comprisefrom about 0.01 μM to about 10 mM of the composition, or from about 10⁻⁴% to about 1% by weight of the composition. More preferably, the activeagent comprises from about 10⁻⁴ % to about 0.1% by weight of thecomposition. More preferably, the active agent comprises from about 10⁻³% to about 0.1% by weight of the composition. In one version of theinvention, the yohimbine, or yohimbine derivative or stereoisomer may beprovided as an extract from the bark of the Johimbe tree (Yohimbe)(Coryuanthe johimbe, or related trees) or from any other natural sourceknown to contain yohimbine or said derivatives or stereoisomers such as(e.g., Rauwolfia serpentina root). Methods of preparing such an extractare known to those of ordinary skill, and may be prepared byhomogenizing the bark in ethanol, then centrifuging the homogenate toremove suspended particles. The supernate may then be combined carrierfor topical application. A more detailed example of an extractionprocess is described below.

The composition may further comprise a sunscreening agent for inhibitingthe melanogenesis induced by ultraviolet light. Such screening agents,or sunscreens, are well known to those of ordinary skill in the art. Asnoted above, the composition comprises a pharmaceutically acceptabletopical carrier capable of delivering the active agent to the skin layercontaining melanocytes under in vivo conditions.

The melanin-decreasing agent (i.e., active agent) is present in thecompositions of the present invention in any effective amount. An"effective amount" of the melanin-decreasing agent is an amount whichdecreases melanogenesis in the treated area of the subject. This amountmay vary with, among other things, the identity of melanin-decreasingagent and carrier, the subject's skin color and condition, and thedegree of depigmentation (i.e., lightening) sought.

The composition of matter of the present invention is preferably applieddirectly to the skin of the individual seeking lightening of the skin.The treated area can be the entire skin surface of the subject or onlythose areas in need of lightening or depigmentation. Application of thecomposition must be repeated periodically to maintain the lightenedcondition of the skin.

As noted above, the compositions of matter of the present inventionpreferably additionally comprises an effective amount of apharmaceutically acceptable topical carrier capable of delivering themelanin-decreasing agent to the melanocyte skin layer under in vivoconditions. The carrier may comprise any solution, suspension, emulsionor any other form which is capable of delivering the agent to themelanocyte skin layer under in vivo conditions. "Capable of delivery",as used herein, means that the carrier should aid the agent in crossingthe stratum corneum and successive cell layers found epidermal to themelanocyte, and/or aids the agent in reaching the layer of cellscontaining melanocytes. Preferably, the carrier should not substantiallyinteract with the agent so that the agent may perform its function asdescribed herein.

The identity and quantity of the carrier will depend on the identity ofthe melanin-decreasing agent used in the composition of the presentinvention. However, in may instances, the carrier will represent fromabout 50% to about 99% of the composition. Preferably the carrier willcomprise an alcohol. Alternatively, the carrier may be liposomes orhydrated lipidic lamellar phases, such as are well-known to those ofordinary skill in the art.

Preferred formulations of the carrier contain an alcohol (e.g.,methanol, ethanol or isopropanol), and a thickener such as propyleneglycol, polyethylene glycol (PEG) or carbopol and a penetration enhancersuch as transcutol.

Specific examples of preferred carrier formulations into which theactive agent is disposed are:

(1) 30% propylene glycol (PG): 70% ethanol;

(2) 30% PG: 1.0-2.5% oleic acid or oleyl alcohol: ethanol (QS);

(3) 10-25% ethoxydiglycol: 0-2.5% oleic acid or oleyl alcohol: 0-5%hydroxypropyl cellulose: ethanol (QS);

(4) 10-50% methylpyrrolidone: 0-20% ethoxydiglycol: 0-2.5% oleic acid oroleyl alcohol: 0-5% hydroxypropyl cellulose: ethanol (QS).

The formulations described herein could be topically applied in a numberof ways to achieve penetration of the active substance through the skinleading to skin lightening. The composition of matter is preferably in agel, lotion or solution form which may be manually rubbed on the skin.Other means of application are acceptable such as aerosol sprays or theuse of an applicator bottle. Typically, the formulation will be appliedover the skin in a dosage of 0.3 to 1 ml/100 cm². This formulation maybe applied at a frequency of every one to two to six to eight to 12hours to the areas where lightening is desired. As defined herein theterm dosage means the amount and frequency of application of theformulation to the skin. A formulation may comprise a film former suchas polyvinyl pyrrolidone or polymethylpyrrolidone. Furthermore, apenetration enhancer (such as transcutol (ethoxydiglycol)) that allowsthe composition to accumulate in the stratum corneum could be used.

Additional ingredients may also be included in the compositions of thepresent invention. Penetration enhancers, when present, will preferablycomprise from about 0.5-25% weight to the solvent mixture and morepreferably will comprise from 1.0-20% by weight. Transcutol is apreferred penetration enhancer, but other known enhancers such as Azone(laurocapram), calcium thioglycolate, alkanecarboxylic acids, liposomes,DMSO, polar lipids, dimethylformamide, N-methyl-2-pyrrolidone, oleicacid, oleyl alcohol, decylmethyl sulfoxide, and propylene glycol arealso enabled herein and may comprise portions of the formulation. Otherpossible penetration enhancers which may be used in any of thecompositions described herein include: lauryl alcohol, dibutyl sebacate,diethyleneglycol oleate, diethyl sebacate, diethyl succinate,diisopropyl sebacate, dioctyl adipate, dioctyl azelate, dioctylsebacate, ethyl acetate, glycerol monolaurate, glycerol monooleate,isopropyl isostearate, isopropyl myristate, isopropyl palmitate, sucrosemonolaurate, sucrose monooleate, lactic acid, lauric acid, linoleicacid, linolenic acid, vaccenic acid, EO-2-oleyl ether, EO-5-oleyl ether,EO-10-oleyl ether, N-methyl-2-pyrrolidone, pyrrolidone/carboxylic acidcombinations, ethanol, polyethylene glycol, Tween 20 and Tween 80.

The composition may further comprise one or more hydrotropic substanceswhich function to increase disorder in the lamellar liquid crystallinestructure of the stratum corneum and thus allow increased transdermaltransport. Examples of such hydrotropes are isopropyl alcohol, propyleneglycol and sodium xylene sulfonate.

Applicants further contemplate that the formulations may comprisecombinations of effective skin lighteners.

The compositions of the present invention may further comprise othercosmetically and therapeutically acceptable carriers or vehiclescontaining other solvents, moisturizers, humectants, oils, emulsifiers,thickeners, thinners, surface active agents, fragrances, preservatives,antioxidants, vitamins and minerals.

While the invention will now be described in connection with certainpreferred embodiments in the following examples so that aspects thereofmay be more fully understood and appreciated, it is not intended tolimit the invention to these particular embodiments. On the contrary, itis intended to cover all alternatives, modifications and equivalents asmay be included within the scope of the invention as defined by theappended claims. Thus the following examples which include preferredembodiments will serve to illustrate the practice of this invention, itbeing understood that the particulars shown are by way of example andfor purposes of illustrative discussion of preferred embodiments of thepresent invention only and are presented in the cause of providing whatis believed to be the most useful and readily understood description offormulation procedures as well as of the principles and conceptualaspects of the invention.

Methodology

Organ Culture System

Use of a human organ culture emulates the human skin in vivo and haspermitted the adequate evaluation and development of the compositionsand methods of treatment of the present invention. The human organculture of the present invention employs a viable foreskin. "Viable"means there has been no substantial morphological change in the foreskinafter surgical removal. Viability may be determined by changes in tissueultrastructure determined through histochemical staining and/or dopareaction staining, techniques which permit monitoring of any changes inthe tissue ultrastructure.

The foreskins may be obtained by circumcising male neonates by standardsurgical procedures. After surgical removal, the foreskin is preferablyprepared for the organ culture by injection intradermally with themedium described hereafter. This swells the mucous membrane and allowsfor the removal of the membrane thereby allowing adequate nutrient flowto the foreskin through the dermis.

The foreskin comprises an epidermis which is normally exposed to theenvironment and a dermis opposing the epidermis. After surgical removaland preparation for the organ culture, the dermis, which is normallysupplied nutrients by the body, is exposed to the environment. In orderto maintain the viability of the foreskin, a nutrient medium suppliesnutrients to the foreskin through the dermis as described hereafter.

The nutrient medium is any composition which maintains the viability ofthe foreskin. Preferably, the nutrient medium has a liquid phase such asa solution, suspension or emulsion. A portion of the medium may beobtained commercially, such as Iscove's modified Dulbecco's medium(IMDM), Ham's nutrient mixture F-10 medium, Minimum essential media(MEM), RPMI media 1630 or 1640, Dulbecco's Modified Eagle Media (D-MEM)or Media 199 all of which are manufactured by Gibco Laboratories ofGrand Island, N.Y. as well as other companies, the specification sheetsof which are hereby incorporated by reference. Additionally the mediumcomprises about 10% to about 30% horse serum and about 2% to about 10%fetal bovine serum; such serums may be purchased from Hyclone Lab Inc.,of Logan, Utah, for example. If necessary, an alkalizer such as sodiumbicarbonate may be added until the medium achieves a preferred pH,preferably about a physiological pH. Antibiotics such as penicillinand/or streptomycin may also be added for microbial control.

If transportation of the foreskin is necessary after surgical removal,the foreskin is immediately placed on an absorbent support saturatedwith the nutrient medium. In order to maintain the viability of theforeskin, the foreskin is disposed in the medium within about 3-4 hoursafter surgical removal. The position of the foreskin in the mediumshould be that the dermis contacts the medium and the epidermis is notsubstantially contacted by the medium.

A complete description of the foreskin organ culture system is found inU.S. Pat. No. 5,540,914 which is hereby incorporated herein by referencein its entirety.

Once the foreskin is disposed in the organ culture system as describedherein, the organ culture system is incubated during the length ofobservation of the foreskin, for example, for 27-72 hours. Preferablythe medium is changed daily, since nutrients may be depleted over time,and the incubation causes degradation of medium components.

In preparing the human organ culture system described herein, theforeskin is surgically removed, prepared and disposed in the organculture system as described herein. Before positioning the foreskin inthe system, the foreskin should be observed to determine the amountand/or the condition of the biological factor under study to obtain abaseline measurement. After treatment of the foreskin with an agent, thebiological factor is again observed for a post-treatment measurement tobe compared to the baseline measurement. For example, if the amount oftyrosinase in the foreskin is under study, the amount of tyrosinase isdetermined as a baseline measurement prior to application of the agentto the foreskin.

If the foreskin is to be treated with an agent under study, the agentcan be added to the nutrient medium so that it comes into contact withthe foreskin through the medium. Alternatively, the agent can be placeddirectly upon the epidermis of the foreskin. The treatment time willdepend on the results sought, the identity of the agent under study, thetime over which the foreskin can remain viable, and other variables.

After the foreskin has been treated with the agent under study, theforeskin may be observed and/or tested in any manner which willdetermine the differences in the foreskin from the observation and/ortests on the untreated foreskin. For example, the activity of tyrosinasemay be measured as described herein and correlated to a decrease inmelanogenesis; the rate of DNA synthesis can be measured by ³H-thymidine uptake and compared to controls; or the increase or decreasein the synthesis of proteins and/or RNA can be measured by determiningthe rate of incorporation of ³ H! leucine (protein) or ³ H! uridine(RNA) into acid-precipitating material. Alternatively, histologicalsections of the treated foreskin can be assessed microscopically asdescribed elsewhere herein.

Preparation of Human Foreskin and Organ Culture

At the time of surgical removal, human foreskins were placed on sterilegauze saturated with sterile IMDM medium (Iscove's modified Dulbecco'smedium purchased from Irvine Scientific of Santa Ana, Calif.) fortransportation from the Hospital nursery to the laboratory. The tissueswere rinsed in sterile IMDM medium containing 500 U/ml penicillin and500 μg/ml of streptomycin for 5 minutes. Under sterile conditions, anintradermal injection of medium was performed from the dermal side priorto dissection of the mucous membrane and lower dermis by scissors tomake the thickness of skins equal. The foreskins were then cut intoapproximately 3 mm×3 mm squares and either frozen at -75° C. or placedin organ culture as described below.

The organ culture medium was prepared from IMDM with glutaminesupplemented with 20% horse serum, 5% fetal bovine serum, 100 U/mlpenicillin, 100 μg/ml streptomycin and 3 mg/ml sodium bicarbonate. Theserums were obtained from Hyclone Lab. Inc. of Logan Utah. Culture unitswere prepared by placing sterilized filters (AP20 025 00, Millipore)over sterilized support screens (25 cm Swinnex filter support screens,Millipore) in the wells of 6-well tissue culture plates (Falcon 3046)with medium added to the wells such that the skin support screensfloated and the filter absorbed the medium from beneath. The tissuesamples were placed, epidermis up, on top of the saturated filters andincubated at 37° C. in a 5% CO₂ humidified atmosphere. The medium waschanged everyday. Harvested cultures were frozen at -75° C.

For histological study (light microscopy), thawed samples of freshtissue and explants were mounted in OCT compound (ICN ImmunoBiologicals, Lisle, Ill.) and frozen by liquid nitrogen. Cryostatsections (6 μm thick) were fixed in 2% formaldehyde for 2 hours at 40°C., and then stained either with hematoxylin and eosin or subjected todopa staining. The dopa reactions were carried out by incubation in twochanges of 0.1% L-dopa solution buffered to pH 7.4 in 0.1M sodiumphosphate buffer for 4 hours at 37° C.

Determination of Tyrosinase Activity

Tyrosinase activity in human skin organ cultures was determined bymeasuring the tyrosine hydroxylase activity of the enzyme. The assaymeasures the production of ³ H₂ O during the conversion of ³ H!tyrosineto L-DOPA. Weighed skin preparations were incubated in 0.3 ml of areaction mixture containing 0.01 mM of L-tyrosine, 5-6 uCi/ml of ³H!tyrosine and 0.1 mM L-DOPA in 0.1 M of pH 6.8 phosphate buffer for 4hours at 37° C. To terminate the reaction, 1 ml of phosphate buffer wasadded, the tubes vortexed, and 0.4 ml aliquots removed in triplicate andmixed with an equal volume of Norit SG activated charcoal (10% w/v, in0.1N HCl). Following centrifugation at 2000×g for 10 min, thesupernatants (0.5 ml) were placed in scintillation vials, scintillationfluid added, and vials counted in a TM Analytic 6895 scintillationcounter equipped with a DPM processor.

Melanocyte Bioassay

The normal human melanocyte cell strains used in this study were derivedfrom foreskins of either neonates or from 2-6 year old black or whitemales. Human melanocyte cultures were grown in Ham's F-10 nutrientmedium supplemented with 10% horse serum, 5% fetal bovine serum (FBS),32 nM TPA (12-O-tetradecanoylphorbol 13-acetate), penicillin (100units/ml), and streptomycin (100 μg/ml).

To determine tyrosinase activity in situ in human melanocyte cultures,the tyrosine hydroxylase activity of the enzyme was determined. Cellswere seeded into 60-mm culture dishes at 2×10⁵ cells/dish and allowed toattach overnight. The medium was then exchanged with a growth mediumcomprising Ham's F-10 nutrient medium +10% FBS+2 μg/ml Bovine PituitaryExtract (BPE)+2 ng/ml of Fibroblast growth factor (FGF), supplementedwith 1 μCi/ml of ³ H!tyrosine (L-ring-3,5-³ H) -tyrosine, DuPont NewEngland Nuclear), and with prostaglandin, where indicated. Cells weregrown in labeled medium for 72 hours, (unless otherwise indicated) andat this time, the medium was removed and assayed for the presence of ³H₂ O using the charcoal absorption method of Pomerantz. Tyrosinaseactivity in cell homogenates was determined by sonicating cell pelletsin 0.1-M sodium phosphate buffer (pH 6.8) and then incubating 50-μlaliquots in 0.5 ml of a reaction mixture containing 0.1-mM tyrosine, 2μCi/ml of ³ H!tyrosine, 0.1-mM L-DOPA (dihydroxyphenylalanine), and 0.1mM PMSF (phenylmethylsulfonyl fluoride) at 37° C. for 2 hours. Reactionswere terminated by the addition of 1 ml of charcoal (10% w/v in 0.1-NHCl). Samples were centrifuged, and the supernatants removed fordetermination of the amount of ³ H₂ O produced.

The amount of melanin in melanocytes was determined by incubating cellpellets in 2 ml of 1-N NaOH for 48 hours at 37° C. and then measuringthe solubilized melanin at 400 nm.

Results

The effects of yohimbine, its stereoisomers and several other compoundson depigmentation in melanocytes and in cultured foreskin are shown inFIGS. 1-8. Melanocytes and foreskins were cultured as described above.

The effects of yohimbine HCl on tyrosinase activity in black melanocytes(strain B415) after a 72 hour incubation period in McX-TPA media areshown in FIG. 1. The results demonstrate a dose response wherein 100 μMyohimbine HCl strongly inhibits melanogenesis in black melanocytes whilethe effect progressively decreases until, at a concentration of 1 μM,the inhibitory effect has virtually disappeared.

FIG. 2 shows the same inhibitory effect of separate batches of yohimbineprepared as a free base (dissolved in ethanol) at a concentration of 100μM. The degree of inhibition is similar to that shown in FIG. 1.

FIG. 3 shows a similar inhibitory effect, at a similar degree, using theyohimbine stereoisomer rauwolscine (α-yohimbine) in white melanocytes(strain W442) incubated for 72 hours.

FIG. 4 shows a pixel graph and a photograph of histological slides ofblack foreskins cultured as described above which have been incubatedfor 24 hours with yohimbine (100 μM) and without yohimbine (the control)in the growth medium. The skin treated with yohimbine is clearly lessdensely stained than the untreated foreskin. In terms of relative pixelvalue, the control foreskin is approximately 5× darker than the foreskinwhich was treated with yohimbine.

FIG. 5 shows the recovery effect in black melanocytes (strain B417).Melanocytes which had been treated with yohimbine (100 μM) were providedwith nutrient media without yohimbine. Within 48 hours, the melaninproduction in the treated melanocytes was back to the same level as themelanocytes exposed to the medium without yohimbine. The resultsdemonstrate that the effect of yohimbine is reversible and that theyohimbine must be repeatedly applied to maintain the inhibitory effect.

FIG. 6 shows an inhibitory effect of two effective α₂ -antagonists BU224and efaroxan on white melanocytes (strain W442) treated for 72 hours.BU224 and efaroxan at concentrations of 100 μM inhibited melanogenesis.

FIG. 7 shows an inhibitory effect of the α-antagonist phentolamine andof the yohimbine stereoisomer corynanthine on white melanocytes (strainW424) treated for 72 hours. Phentolamine and corynanthine at 100 μMinhibited melanogenesis.

FIG. 8 shows a pixel graph and a photograph of histological slides ofblack foreskins cultured as described above, which were incubated for 24hours with and without a yohimbine-containing extract of Johimbe bark(extraction process described below). The skin treated with theyohimbine-containing extract of Johimbe bark is clearly less denselystained than the ethanol control. In terms of relative density, thecontrol foreskin is approximately 4× darker than foreskin which wastreated with the extract.

Johimbe Bark Extraction Method

Ten g of Johimbe (Yohimbe) bark (obtainable commercially, for example,from the Gaia Herb Co.) was milled in a coffee mill for 20 seconds toreduce bark to fine sediment, and then 100 ml of water was added. Themixture was stirred for 1 hour at room temperature in a 250 mlErlenmeyer flask and then centrifuged at 5000×g for 20 minutes. Thesupernatant was discarded and the sediment containing yohimbine wasresuspended in 100 ml of ethanol. This mixture was sonicated briefly andthen stirred for 5 hours at room temperature to further extract theyohimbine from the bark. After stirring, the mixture was againcentrifuged and the supernatant saved. The pellet was discarded. Theethanol supernatant was evaporated under nitrogen gas until the volumehad been reduced to 16 ml. This resulted in an ethanol solution whichcontained approximately 100 microMolar yohimbine. The concentration ofyohimbine in the extract will vary depending on the bark source andtechnique used.

It will be understood by a person of ordinary skill in the art that theextraction method described herein is but one of many suitable methodswhich may be used to obtain an extract of yohimbine and all such methodsare contemplated as being within the scope of the present invention asclaimed herein.

These data show that yohimbine and various derivatives and stereoisomersthereof and certain effective α₂ -antagonists are effective indecreasing tyrosinase activity in human melanocyte cells therebyinhibiting melanogenesis leading to skin lightening.

Changes may be made in the construction and the operation of the variouscompositions described herein or in the steps or the sequence of stepsof the methods described herein without departing from the spirit andscope of the invention as defined in the following claims.

What is claimed is:
 1. A method of causing lightening of the epidermis,comprising:providing a composition comprising: an active agentcomprising an amount of at least one of yohimbine, rauwolscine,corynanthine or allo-yohimbine or a stereoisomer, or salt thereof whichis effective in inhibiting melanogenesis in skin melanocytes, and acosmetically or pharmaceutically acceptable topical vehicle or carrier;and topically applying the composition to the epidermis in an amountsufficient to at least partially inhibit melanogenesis in the epidermis.2. The method of claim 1 wherein the yohimbine of the composition isprovided as an extract of Johimbe bark.
 3. The method of claim 1 whereinthe carrier or vehicle comprises an alcohol.
 4. The method of claim 1wherein the carrier or vehicle comprises a penetration enhancer.
 5. Themethod of claim 2 wherein said extract comprises from 0.01% to 80% ofthe composition.
 6. The method of claim 1 wherein the active agentcomprises from 0.0001% to 5% of the composition.
 7. A method of causinglightening of the epidermis, comprising:providing a compositioncomprising an extract of Johimbe bark which comprises an active agentcomprising at least one of yohimbine, rauwolscine, corynanthine,allo-yohimbine or a stereoisomer or salt thereof and a cosmetically orpharmaceutically acceptable carrier or vehicle; and topically applyingthe composition to the epidermis in an amount sufficient to at leastpartially inhibit melanogenesis in the epidermis.
 8. The method of claim7 wherein the carrier or vehicle comprises an alcohol.
 9. The method ofclaim 7 wherein the carrier or vehicle comprises a penetration enhancer.10. The method of claim 7 wherein said Johimbe bark extract comprisesfrom 0.01% to 80% of the composition.
 11. The method of claim 7 whereinthe active agent comprises from 0.01% to 5% of the composition.